The gut neuroendocrine system is a key regulator of intestinal secretion, motility and pain, but its role in the symptoms, including secretory and motility abnormalities, mucosal damage, strictures and pain that characterize inflammatory bowel diseases (IBD), particularly Crohn's, has not been well-investigated. We propose that the most prevalent intestinal neuroendocrine cell, the enterochromaffin (EC) cell, plays a critical role in Crohn's symptomatology. The EC cell regulates normal intestinal motility and fluid secretion via secretion of serotonin (fluid and electrolyte secretion/pain), guanylin (enterocyte secretion) and substance P (motility/pain). Known effectors of normal EC cell function include luminal- and strain-sensing and neural (1-adrenergic) activation. The studies in this proposal will examine how inflammation-related products and mechanical stimulation activate NF?? and ERK signaling in EC cells and augment the secretory and motility symptomatology that characterizes Crohn's. We hypothesize that altered Crohn's EC cell activation and/or function are due to inflammatory-mediated events on the EC cell: either increased responsiveness to physiological 1-adrenergic- and strain mediated serotonin synthesis and secretion, or a loss of normal inhibitory responses to somatostatin and acetylcholine. Cytokines (particularly IL1) and bacterial lipopolysaccharides (LPS) and muramyl dipeptide (MDP) have been implicated in Crohn's pathogenesis through activation of TOLL4 receptor and IL1 receptor (TIL) and NOD2/CARD15 signaling. We propose that this system plays a pivotal role in abnormal Crohn's EC cell function via activation of signaling through NF??, and/or ERK pathways. The functional consequence would be an increase in transcription of the rate-limiting serotonin synthesizing enzyme, tryptophan hydroxylase (Tph-1), with a resultant increase in serotonin synthesis and secretion. Excess of serotonin and co-secreted products, guanylin and substance P, would produce increased secretion, motility and pain - all hallmarks of Crohn's disease. Additionally, loss of inhibitory regulation by somatostatin or acetylcholine might amplify these effects. In preliminary data, using FACS sorting, we produced pure (>99%), fully functional human and mouse (IL10R knockout) intestinal EC cells from normal and colitis mucosa. Using real-time PCR, we identified 5-fold increased transcripts for TLR4, 10-fold for IL1R, 2-fold for somatostatin receptors (1-3, 5) and 2-fold for the stimulatory mechano-responsive G-protein coupled receptor ADORA2B in human Crohn's EC cells. Both E. coli LPS and IL1 significantly increased 5HT secretion >2-fold in Crohn's EC cells. The NF?? and ERK phosphorylation signaled secretory responses were reversed by LPS and IL1 receptor antagonists (K12 LPS and IL1RA) and inhibited (80%) by a somatostatin analog in human samples. Cyclic strain application to EC cells resulted in 5HT release, induction of substance P and guanylin transcription in a time-dependent fashion. In the murine model, mucosal 5HT levels and Tph-1 transcripts were increased in colitis, TLR4 and IL1R were expressed in colitis EC cells and both E. coli LPS and IL1 significantly increased 5HT secretion >3-fold. These results indicate that EC cell function is altered in Crohn's disease and that inflammatory mediators and mechanical strain play a direct role in increasing 5HT secretion. We propose to establish how EC cell activity is altered in Crohn's by: 1) determining whether 5HT, substance P and guanylin secretion is regulated by norepinephrine, mechanic forces somatostatin analogs and acetylcholine in Crohn's EC cells; 2) determining if 1-adrenergic and strain-mediated activation (cAMP signaling and PKA activation) is increased in Crohn's EC cells; 3) identifying whether the principal TIL/NOD2/CARD15 signaling pathway components, NF?? and ERK, are over-expressed in Crohn's EC cells and responsible for the elevated 5HT synthesis and secretion; and 4) Analyze EC cell function in a murine colitis model and evaluate whether inhibiting EC cell secretion ameliorates colitis symptoms. Crohn's is a common and chronic disease that substantially reduces quality of life. The understanding of the pathophysiology behind its often devastating symptomatology remains poorly understood. The identification and delineation of abnormal regulation of neuroendocrine cell function by the inflammatory response would provide novel information regarding the pathogenesis of Crohn's disease. In addition, it may provide the potential for novel therapeutic targets in Crohn's disease and possibly other forms of inflammatory bowel disease.